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Control of Microbial Growth

Commerical Sterilization

Sterilization: removal (destruction) of all forms of microbial life
- including endospores, but with the possible exception of prions, they have a tendency to be able to survive
- usually done byb steam under pressure or a sterilizing gas, such as ethylene oxide

Commercial Sterilization: using heat (to destroy) microbes including some endospore formers in canned food
- more resistant endospores of thermophilic bacteria may survive
- i.e. commercial: C. botulinum in autoclave

Degerming: mechanical cleansing to remove microbes, rather than killing, from a limited area
- i.e. alcohol soaked swab prior to injection
- mostly mechanical removal

Antiseptic: non-toxic chemicals that kill most microbes
- i.e. over the counter hand soaps

Definitions Cont:
Aseptic Technique

Disinfectant: toxic chemical (to tissue) that kills microbes (excludes endospore formers)
- destruction of vegetative pathogens, may make use of mechanical or chemical methods
- directed at destroying harmful MO's
- i.e. Lysol

Aseptic technique: without infection, implies technique used to prevent entry of microbes
- i.e. disinfecting lab station, flaming loop and tube top

Asepsis: without infection

Bacteriostatic: merely inhibiting growth & multiplication of microbes

Bacteriocidal: kills microbes

Effective Disinfection is Dependent on
Bacterial Type
Rapid Acting
Hampered by Matter
Corrosion Factor

Bacterial type:
- LPS (lipo-poly-saccharide) - part of the gram negative cell wall therefore more resistant
- Endospores - resistant to disinfectant
- viral-prions - more resistant to disinfectant

Rapid Acting: if disinfectant acts more rapidly, its more effective

Hampered by Matter: if disinfectant is not hampered by organic matter (blood, body fluids)

Stability: if disinfectant is stable

Corrosion Factor: it does not weaken or corrode, maintains effectiveness over time

Rate of Microbial Death

- microbes die at constant rate (p.189)
- there is a logarithmic decrease in death rate, when plotted, is a straight line
- for every minute of treatment, there is a 90% reduction rate of bacteria when chemically treated or heated
- factors that can impact effectiveness of antimicrobial treatment include:
- concentration of bacteria
- types (Genus) of microbe
- temperature of medium
- how does one measure the effectiveness of an antimicrobial agent? Disk-Diffusion Method (aka Kirby-Bauer Method)

Disk Diffusion Method
Also Called....
Media (agar)
Standard Criteria
Def: In-Virto

- also called Kirby-Bauer Method
- media (agar): use Mueller Hinton Agar
- standardized criteria: pH 7.2-7.6,
- depth 4mm, sterile in 150mm Petri dish
- disks: have chemical (or drug) impregnated onto disk
- disk stored refridgerated
- disk is placed on a plate containing agar gel which has been inoculated and then incubated
- a suspension needs to be made (using a broth) of 0.5 McF.
- plate is read for zone of inhibition (p.198-199)
- in-vitro: in the lab

Disk Diffusion Method Cont
Measuring Zone
How much if effective?
Resistant, Sensitive, Intermediate (Size of zone)
Def: in-vivo

- if zone is present it is measured (mm) to see if it is large enough zone
- ≥ 18mm zone of inhibition then drug is effective
- if so, then test chemical (drug) is effective!
- 1: penicillin - R
- 2: ampicillin - R
- 3: cipro - S
- 4: tetra - I
- 1, 2 --> bacteria is resistant to zone 1, 2
- 3 = 28mm inhibited, bacteria are sensitive to 3
- 4 = 13mm, bacteria are intermediate (maybe inhibited)
- Might be able to be used against infection
- Would want to monitor what is going on invivo

Physical Methods:
Direct flame/Incineration

Boiling: kills vegetative forms of bacterial pathogens, almost all viruses & fungi, but less effective on endospores, protein structure is denatured
- how does it inhibit? It denatures the microbe

Autoclaving: steaming under high temperature and pressure. Will sterilize most microbes, protein denatured via moist heat (parameters: 15 psi, 121ºC, 15 min.)
- prefered method of sterilization unless the materials can be damaged by heat or moisture

Direct flame / incineration: burning, dry heat sterilization kills by oxidation

Pasteurization: lowers total bacterial count while preserving flavor of food, denatures bacterial protein (72ºC for 15 sec.) preserves flavor since done so quickly
- known as HTST (hugh-tempurature short-time) pasteurization

- Ionizing: gamma rays (x-rays), forms hydroxyl radicals, good penetration (down to bone)
- Non-Ionizing: utilizes UV light, is bacteriocidal, causes mutation of bacterial DNA, poor penetration (good for soft tissue infections)

Physical Methods Cont:
Filter Sterilization
Culture Medium

Refridgeration: is bacteriostatic, slows bacterial metabolism
- decreased chemical reactions & possible changes in proteins

Filter sterilization: is useful for sterilizing liquids that are destroyed by heat (autoclaving) (i.e. vaccines)
- seperation of bacteria from suspending liquid

Antibiotic: inhibit the growth of certain endospore-forming spoilage bacteria


Culture medium

Chemical Methods:

Phenol (Lister): disruption of plasma membrane, denaturation of enzymes
- rarely used, except as a standard of comparison
- iritates skin & had a disagreeable odor

Phenolics: disruption of plasma membrane, denaturation of enzymes
- environmental surfaces, instruments, skin surfaces, and mucous membranes (pus, saliva, feces)
- derivative of phenol, contain a molecule of phenol that has been chemically altered to reduce its irritating qualities
- remain active in the presence or organic compounds, are stable, and persist for long periods
- most commonly used: O-phenylphenol, main ingredient in Lysol

Biguanides: disruption of plasma membrane
- skin disinfectant, especially for surgical scrubs & pre-op skin preparation in patients
- effective against gram pos bacteria & most gram neg bacteria
- best known biguanide is chlorhexidine, frequently used for MO control on skin & mucous membranes

Alcohols: protein denaturation and lipid dissolution
- thermometers and other instruments, in swabbing the skin with alcohol before an injection
- effectively kill bacteria and fungi, but not endospores and nonevelope viruses
- unsatisfactory when applied to wounds, they cause coagulation of a layer of protein under which bacteria continue to grow
- most commonly used: ethanol & isopropanol

Cemical Methods Cont.

Halogens: iodine inhibits protein function and is a strong oxidizing agent, & chlorine forms the strong oxidizing agent hypochlorous acid which it alters cellular components
- iodine is an effective antiseptic as a tincture and an iodophor, skin disinfection & wound treatment; chlorine gas is used to disinfect water, chlorine compounds are used to disinfect dairy equipment, eating utensils, household items, and glassware

Aldehydes: protein denaturation, used by morticians for embalming
- glutaraldehyde is less irritating than formaldehyde and is used for disinfection of medical equipment, one of the most effective chemical disinfectants
- formaldehyde gas is an excelllent disinfectant, most easily available in the form of formalin which was once used to preserve biological specimens and inactive bacteria and viruses in vaccines.
- glutaraldehyde is less irritating than formaldehyde and more effective. One of the few liquid chemical disinfectants that can be considered a sterilizing agent

Peroxygens: oxidation
- contaminated surfaces, some deep wounds, in which they are very effective against oxygen-sensitive anaerobes

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